Phosphorylation is one of the most common post-translational modifications (PTM) of proteins (approximately 30% of cellular proteins are phosphorylated), and it plays an important role in a wide range of biological processes, such as signal transduction. Phosphorylation is the addition of a phosphate (PO43−) group to a protein or other organic molecule. Phosphorylation turns many protein enzymes on and off, thereby altering their function and activity. Protein phosphorylation in particular plays a significant role in a wide range of cellular processes. Its prominent role in biochemistry is the subject of a very large body of research.
Mass spectrometry (MS) has become an increasingly viable technology for phosphoprotein analysis. However, a major challenge in this regard is that phosphopeptide signal in the positive ion mode is severely suppressed by non-phosphorylated peptides when a phosphoprotein digest is ionized by traditional ionization methods such as electrospray ionization (ESI) in a commonly used “bottom-up” approach. Preliminary purification of phosphopeptides prior to MS analysis is often indispensable to solve the problem, using antibodies, affinity chromatography, metal oxides, nanopolymers, or nanoparticles to enrich phosphopeptides. As separation and purification could be time-consuming, a direct, rapid and sensitive method for ionizing phosphopeptides in mixtures would be instrumental to facilitating their analysis and characterization.
Desorption electrospray ionization (DESI) is a recent advance in the field of MS. DESI provides direct ionization of analytes with little or no sample preparation. Sample ionization by DESI occurs via the interactions with charged microdroplets generated in a pneumatically assisted electrospray of an appropriate solvent. In addition to analysis of solid samples, DESI has been extended to directly ionize liquid samples, and its demonstrated applications include the coupling MS with chromatography, microfluidics, and electrochemistry, probing protein conformation, and developing submilli-second time-resolved MS.